| Product Description | Aladdin's Acid Alcohol Fast Differentiation Solution is generally used in hematoxylin staining, hematoxylin-eosin staining (HE staining) or Masson staining to reduce the staining background from cytoplasm, making the hematoxylin stain of nucleus more lucid.This product developed by aladdin requires about 10 seconds for sample differentiation, enabling a convenient control of the differentiation. If a faster differentiation is desired, we recommend using the Acid Alcohol Superfast Differentiation Solution . The Acid Alcohol Slow Differentiation Solution is also available if a slower differentiation is required.A better hematoxylin stainng result can be obtained if the differentiation step is performed. The Acid Alcohol Differentiation Solution reduces the stain background effectively by removing the hematoxylin stain outside nucleus. Meanwhile, it also weakens the over-stained area in nucleus to improve the hematoxylin staining result of nucleus.Hematoxylin staining is one of the most commonly used methods for staining tissues and cells. The colorless hematoxylin is oxidized to form hematein with a quinoid ring structure, which can form a colored positively charged complex with trivalent aluminum ions, iron ions, etc. In the DNA double helix in nucleus, the phosphate groups on double strands are outward, negatively charged and acidic, and easily bind with the positively charged hematein complex to stain nucleus in purple. Hematoxylin Staining Solution prepared in different ways can stain cell nuclei in blue or blue-violet with different intensities.Hematoxylin is an excellent dye for staining nucleus. However, unexpected cytoplasm staining generally occurs due to the presence of RNA and mitochondrial DNA in cytoplasm, resulting in a high cytoplasm stain background or an over-staining of nucleus. The function of the Acid Alcohol Differentiation Solution is to weaken the colored complex of oxidized hematoxylin and RNA or DNA through the pH value of the solution, so that there is almost no hematoxylin staining in the cytoplasm while the hematoxylin staining of nucleus is relatively moderate. During the differentiation process, the hematoxylin stain in nucleus appears red under acidic conditions. When nucleus staining reaches an appropriate level, the differentiation is stopped immediately by washing samples with tap water and the hematoxylin stain in nucleus returns blue. The appropriate differentiation means that nucleus membrane and chromatin in nucleus especially have clear morphology while the cytoplasm and other parts have basically no background staining. Cytoplasm can be stained in red in contrast by the subsequent eosin staining. Insufficient or over differentiation should be avoided by adjusting the differentiation time properly.The formula of this product has been optimized to have a relatively faster differentiation, compared to the Acid Alcohol Slow Differentiation Solution . This product requires about 10 seconds for the differentiation. It is suitable for researchers who are not familiar with the differentiation procedures. The hematoxylin staining result is improved significantly by the addition of differentiation step. Please see Figure 1 for the hematoxylin staining result before or after differentiation by the Acid Alcohol Fast Differentiation Solution . Figure 1. Paraffin sections of mouse testis stained with hematoxylin and observed before differentiation (Left) or after differentiation (Right) by the Acid Alcohol Fast Differentiation Solution . Sample after differentiation (Right) has reduced background from cytoplasm, and clearer nuclei, compared to the sample before differentiation (Left). Scale bar, 100μm. This figure is for reference only, the actual results may vary due to different experimental conditions. When 0.2ml of Differentiation Solution is used for each sample, C0163S, C0163M and C0163L products are sufficient for the differentiation of 500, 2500 and 10,000 samples, respectively. When using the immersion method for differentiation, the Differentiation Solution can be resued for 10-20 times until its performance decreases significantly.
Precautions： This product is volatile. Please keep the vial tightly closed after use. The reagent should be used up as soon as possible after opening.The differentiation step after staining is optional, but if the differentiation step is performed, the nucleus staining result will be better with clearer morphological structure of nuclear membrane and chromatin.When there is a large number of samples, staining racks (FJ024, FJ026, FJ030, FJ060) or tanks produced by can be used for easier operation.The C0163L product, the Acid Alcohol Fast Differentiation Solution (20X), needs to be diluted to 1X with 75% ethanol prior to use.The duration period of differentiation should be optimized for different samples based on your experimental requirements. For first-time users of this product, it is recommended to perform a time course experiment to determine the best differentiation time for your samples. The Acid Alcohol Superfast Differentiation Solution and the Acid Alcohol Slow Differentiation Solution are also available for different experimental requirement.This product is volatile and flammable, and must be stored hermetically in a cool, dry and well-ventilated safety cabinet away from open fire, spark, heat source, and hot surface. Smoking is prohibited.This product is corrosive. Please be careful when handling the reagent and reactions, and take effective measures to avoid direct contact or corrosion of other objects.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear lab coat and disposable gloves during the operation. Materials Required But Not Supplieda. Fix Solution: Immunol Staining Fix Solution , or 4% Paraformaldehyde Fix Solution .b. Hematoxylin Staining Solution: Hematoxylin Staining Solution or Hematoxylin and Eosin Staining Kit .c. If dehydration, clearing and mounting are required, xylene and mounting medium can be purchase from , such as Polyvinylpyrrolidone Mounting Medium , Antifade Mounting Medium , neutral gum or other mounting media.d. Different concentrations of ethanol: Absolute ethanol, 90% ethanol, 80% ethanol, 75% ethanol.e. Distilled water.
Instructions for Use： 1. Sample preparation: a. For paraffin sectionsDewax paraffin sections in xylene for 5-10 minutes;Replace with fresh xylene and dewax for another 5-10 minutes;Anhydrous ethanol for 5 minutes;90% ethanol for 2 minutes;80% ethanol for 2 minutes;70% ethanol for 2 minutes;Distilled water for 2 minutes;Proceed to step 2.b. For frozen sectionsFix frozen sections in Fix Solution for more than 10 minutes;Wash twice with distilled water for 2 minutes each;Proceed to step 2.c. For cultured cellsFix cells in Fix Solution for more than 10 minutes;Wash cells twice with distilled water for 2 minutes each;Proceed to step 2.2. Hematoxylin staining:a. Stain samples with Hematoxylin Staining Solution for 5-10 minutes (Time can be adjusted appropriately).b. Rinse samples with tap water to remove the extra staining solution, and immerse samples in tap water for 10 minutes.c. Wash with distilled water once for a few seconds.d. Perform sample differentiation with the Acid Alcohol Fast Differentiation Solution for 10 seconds, and then rinse with tap water for 10 minutes. The differentiation time can be adjusted appropriately.e. Wash samples with 70% ethanol twice, examine cells directly or proceed to eosin staining or sample mounting after dehydration and clearing. Note: For counterstaining of sample that has been subdued to immunohistochemistry staining, the hematoxylin staining can be followed directly after completing immunohistochemistry staining.
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